Proton therapy (PT) is one of the most advanced modalities of radiation therapy. However, uncertainties in the treatment plan have a much higher impact than in photon therapy due to the highly localized dose deposition. Range uncertainties of up to several centimeters can be introduced during treatment planning. Detailed verification of dose delivery would allow the full potential of PT to be realized. PET imaging of radioactivity distributions produced by nuclear fragmentation reactions during treatments has emerged as the only practical approach for in vivo treatment verification. We have used an in-room PET scanner to measure the radioactivity concentration history after proton irradiation, showing that 15O forms more than 80% of the PET signal immediately after irradiation. Accordingly, we have developed a completely new theory which estimates isotope-specific production and clearance rates directly from the data. The isotope production is a fundamental endpoint since it is a unique function of the dose delivered with a given proton profile and tissue composition. The biological clearance rates of 15O can be calculated concurrently, which may carry valuable physiological information about tumor environment. In this project we will develop new methods for in-room dose verification of proton therapy, based on the 15O production rate maps and Dual Energy CT (DECT). DECT can provide more accurate tissue property information, which will improve the Monte Carlo prediction of PET activity distributions, and also can potentially improve the treatment planning accuracy. We propose controlled validation in phantoms, experimental confirmation in animals, and pilot human studies. In-room imaging will be improved with a new mobile PET/CT scanner offering high sensitivity PET and a co-registered DECT. We will also evaluate the feasibility of assessing tumor responses to treatment using 15O biological clearance rates with radiation-sensitive VX2 tumors and in clinical studies, and investigate the value of DECT in more accurate proton therapy treatment planning.